Ca ²⁺ releases E‐Syt1 autoinhibition to couple ER ‐plasma membrane tethering with lipid transport

Abstract

The extended synaptotagmins (E-Syts) are endoplasmic reticulum (ER) proteins that bind the plasma membrane (PM) via C2 domains and transport lipids between them via SMP domains. E-Syt1 tethers and transports lipids in a Ca-dependent manner, but the role of Ca in this regulation is unclear. Of the five C2 domains of E-Syt1, only C2A and C2C contain Ca-binding sites. Using liposome-based assays, we show that Ca binding to C2C promotes E-Syt1-mediated membrane tethering by releasing an inhibition that prevents C2E from interacting with PI(4,5)P-rich membranes, as previously suggested by studies in semi-permeabilized cells. Importantly, Ca binding to C2A enables lipid transport by releasing a charge-based autoinhibitory interaction between this domain and the SMP domain. Supporting these results, E-Syt1 constructs defective in Ca binding in either C2A or C2C failed to rescue two defects in PM lipid homeostasis observed in E-Syts KO cells, delayed diacylglycerol clearance from the PM and impaired Ca-triggered phosphatidylserine scrambling. Thus, a main effect of Ca on E-Syt1 is to reverse an autoinhibited state and to couple membrane tethering with lipid transport.

In Vitro Assays to Measure the Membrane Tethering and Lipid Transport Activities of the Extended Synaptotagmins

“…Work based on these assays validated the hypothesis that E-Syt1 has both membrane tethering and lipid transfer activities and provided insight into the regulation of its activities [5, 10, 22]. These assays can be adapted to explore and/or characterize the membrane tethering and lipid transfer properties of other proteins.…”

“…Plasmids: pCMV6-AN-His vector (e.g., OriGene) containing the region coding for residues 93–1104 of human E-Syt1 ( see [5, 10] for detailed information for making this construct).…”

“…( a ) Domain organization of the E-Syts and of the E-Syt1 construct used for the membrane tethering and lipid transfer assays. ( b ) Schematic representation of the putative arrangement of E-Syt1 at ER-PM contacts in the presence of elevated cytosolic Ca 2+ ( see also [5]). The protein dimerizes via its SMP domain.…”

“…To prove a lipid transfer function of the E-Syts, and gain insight into the regulation of such function as well as into their tethering properties, we used in vitro liposome-based assays and purified fragments of the cytosolic portion of E-Syt1 [5, 10, 22]. Such fragments had a Histidine tag at their N-terminus so that they could be anchored to liposomes containing a nickel-chelating lipid [23].…”

“…We found that Ca 2+ in the micromolar range was required for efficient lipid transfer (Fig. 3a) and that the effect of Ca 2+ was due to the release of E-Syt1 from an autoinhibitory conformation affecting both its membrane tethering properties and accessibility of lipid to its lipid-binding pocket [5]. Dequenching could also be the result of fusion, but this was excluded by control experiments in which sodium dithionite, a poorly membrane permeable compound, was added to liposomes before protein addition to quench NBD fluorescence in their outer leaflet [25].…”